Mitochondrial diseases are devastating disorders for which there is no cure, and no proven treatment. About 1 in 2000 children are born each year who will develop mitochondrial disease in their lifetime. Half of these will show symptoms before age 5, and 80% of these will die before age 20. The other half will develop disease between age 5 and 65. Mortality is roughly that of cancer, and can range between 5% to 50% per year after diagnosis. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much research is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genomes, either as point mutations, deletions, rearrangements, or depletion of the mtDNA. The mutation rate of the mitochondrial genome is 10-20 times greater than in the nuclear DNA and more prone to oxidative damage than nuclear DNA. Mutations in human mitochondrial DNA cause premature aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. We previosly cloned, overexpressed, purified, and characterized the human pol gamma catalytic subunit, p140, and the p55 accessory subunit. We have determined that the accessory subunit functions as a processivity factor and enhances DNA binding of the holoenzyme. We have investigated the fidelity of DNA replication by pol gamma with and without exonucleolytic proofreading and its p55 accessory subunit. Pol gamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Several nuclear genes involved in mitochondrial maintenance have recently been identified as disease loci for mitochondrial disorders. The pol gamma gene and the Twinkle gene (DNA helicase) are two genes that when mutated cause progressive external ophthalmoplegia. Additionally, other mutations in the pol gamma gene are associated with male infertility (causing decrease in sperm motility), Alpers syndrome, sensory ataxia and neuropathy, and Parkinson's disease. We are studying the molecular pathological effects of disease mutations in pol gamma and Twinkle gene products. Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mitochondrial DNA (mtDNA). Mutations in the gene for the pol gamma, POLG, are associated with PEO and other mitochondrial disorders. To date, over forty mutations in the POLG gene in are reported to cause heritable PEO and other mitochondrial disorders, but the functional consequences of these mutations are unknown. Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease. Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol gamma) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE-dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE-dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE-dG adducts but not past BaP or BcPh DE-dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol gamma opposite the BaP DE-dG adducts resembles that observed with the Y family pol eta. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.